Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Jul 23;93(15):7572–7576. doi: 10.1073/pnas.93.15.7572

Expression cloning of the Golgi CMP-sialic acid transporter.

M Eckhardt 1, M Mühlenhoff 1, A Bethe 1, R Gerardy-Schahn 1
PMCID: PMC38787  PMID: 8755516

Abstract

Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.

Full text

PDF
7572

Images in this article

7573Fig. 1
on p.7573
7575Fig. 3
on p.7575
7575Fig. 4
on p.7575

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bierhuizen M. F., Fukuda M. Expression cloning of a cDNA encoding UDP-GlcNAc:Gal beta 1-3-GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9326–9330. doi: 10.1073/pnas.89.19.9326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bloch R. J. Clusters of neural cell adhesion molecule at sites of cell-cell contact. J Cell Biol. 1992 Jan;116(2):449–463. doi: 10.1083/jcb.116.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bretscher M. S., Munro S. Cholesterol and the Golgi apparatus. Science. 1993 Sep 3;261(5126):1280–1281. doi: 10.1126/science.8362242. [DOI] [PubMed] [Google Scholar]
  4. Briles E. B., Li E., Kornfeld S. Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose. J Biol Chem. 1977 Feb 10;252(3):1107–1116. [PubMed] [Google Scholar]
  5. Cacan R., Cecchelli R., Hoflack B., Verbert A. Intralumenal pool and transport of CMP-N-acetylneuraminic acid, GDP-fucose and UDP-galactose. Study with plasma-membrane-permeabilized mouse thymocytes. Biochem J. 1984 Nov 15;224(1):277–284. doi: 10.1042/bj2240277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Capasso J. M., Hirschberg C. B. Effect of nucleotides on translocation of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi apparatus vesicles. Biochim Biophys Acta. 1984 Oct 17;777(1):133–139. doi: 10.1016/0005-2736(84)90505-4. [DOI] [PubMed] [Google Scholar]
  7. Capasso J. M., Hirschberg C. B. Mechanisms of glycosylation and sulfation in the Golgi apparatus: evidence for nucleotide sugar/nucleoside monophosphate and nucleotide sulfate/nucleoside monophosphate antiports in the Golgi apparatus membrane. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7051–7055. doi: 10.1073/pnas.81.22.7051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  9. Chomczynski P., Mackey K. One-hour downward capillary blotting of RNA at neutral pH. Anal Biochem. 1994 Sep;221(2):303–305. doi: 10.1006/abio.1994.1416. [DOI] [PubMed] [Google Scholar]
  10. Cummings L., Warren C. E., Granovsky M., Dennis J. W. Antisense and sense cDNA expression cloning using autonomously replicating vectors and toxic lectin selection. Biochem Biophys Res Commun. 1993 Sep 15;195(2):814–822. doi: 10.1006/bbrc.1993.2118. [DOI] [PubMed] [Google Scholar]
  11. Davidson R. L., Gerald P. S. Induction of mammalian somatic cell hybridization by polyethylene glycol. Methods Cell Biol. 1977;15:325–338. doi: 10.1016/s0091-679x(08)60223-x. [DOI] [PubMed] [Google Scholar]
  12. Deutscher S. L., Hirschberg C. B. Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle membranes. J Biol Chem. 1986 Jan 5;261(1):96–100. [PubMed] [Google Scholar]
  13. Deutscher S. L., Nuwayhid N., Stanley P., Briles E. I., Hirschberg C. B. Translocation across Golgi vesicle membranes: a CHO glycosylation mutant deficient in CMP-sialic acid transport. Cell. 1984 Dec;39(2 Pt 1):295–299. doi: 10.1016/0092-8674(84)90007-2. [DOI] [PubMed] [Google Scholar]
  14. Eckhardt M., Mühlenhoff M., Bethe A., Koopman J., Frosch M., Gerardy-Schahn R. Molecular characterization of eukaryotic polysialyltransferase-1. Nature. 1995 Feb 23;373(6516):715–718. doi: 10.1038/373715a0. [DOI] [PubMed] [Google Scholar]
  15. Fasman G. D., Gilbert W. A. The prediction of transmembrane protein sequences and their conformation: an evaluation. Trends Biochem Sci. 1990 Mar;15(3):89–92. doi: 10.1016/0968-0004(90)90187-g. [DOI] [PubMed] [Google Scholar]
  16. Field M. C., Wainwright L. J. Molecular cloning of eukaryotic glycoprotein and glycolipid glycosyltransferases: a survey. Glycobiology. 1995 Jul;5(5):463–472. doi: 10.1093/glycob/5.5.463. [DOI] [PubMed] [Google Scholar]
  17. Fleischer B., McIntyre J. O., Kempner E. S. Target sizes of galactosyltransferase, sialyltransferase, and uridine diphosphatase in Golgi apparatus of rat liver. Biochemistry. 1993 Mar 2;32(8):2076–2081. doi: 10.1021/bi00059a027. [DOI] [PubMed] [Google Scholar]
  18. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Frosch M., Görgen I., Boulnois G. J., Timmis K. N., Bitter-Suermann D. NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B meningococci. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1194–1198. doi: 10.1073/pnas.82.4.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gerardy-Schahn R., Eckhardt M. Hot spots of antigenicity in the neural cell adhesion molecule NCAM. Int J Cancer Suppl. 1994;8:38–42. doi: 10.1002/ijc.2910570708. [DOI] [PubMed] [Google Scholar]
  21. Gleeson P. A., Teasdale R. D., Burke J. Targeting of proteins to the Golgi apparatus. Glycoconj J. 1994 Oct;11(5):381–394. doi: 10.1007/BF00731273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Harvey B. E., Thomas P. Inhibition of CMP-sialic acid transport in human liver and colorectal cancer cell lines by a sialic acid nucleoside conjugate (KI-8110). Biochem Biophys Res Commun. 1993 Jan 29;190(2):571–575. doi: 10.1006/bbrc.1993.1086. [DOI] [PubMed] [Google Scholar]
  23. Harvey B. E., Toth C. A., Wagner H. E., Steele G. D., Jr, Thomas P. Sialyltransferase activity and hepatic tumor growth in a nude mouse model of colorectal cancer metastases. Cancer Res. 1992 Apr 1;52(7):1775–1779. [PubMed] [Google Scholar]
  24. Hirschberg C. B., Snider M. D. Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem. 1987;56:63–87. doi: 10.1146/annurev.bi.56.070187.000431. [DOI] [PubMed] [Google Scholar]
  25. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  26. Höltke H. J., Kessler C. Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA-based detection. Nucleic Acids Res. 1990 Oct 11;18(19):5843–5851. doi: 10.1093/nar/18.19.5843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kean E. L. Sialic acid activation. Glycobiology. 1991 Nov;1(5):441–447. doi: 10.1093/glycob/1.5.441. [DOI] [PubMed] [Google Scholar]
  28. Klein P., Kanehisa M., DeLisi C. The detection and classification of membrane-spanning proteins. Biochim Biophys Acta. 1985 May 28;815(3):468–476. doi: 10.1016/0005-2736(85)90375-x. [DOI] [PubMed] [Google Scholar]
  29. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  30. Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem. 1991 Oct 25;266(30):19867–19870. [PubMed] [Google Scholar]
  31. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  32. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  35. Leucine-zipper motif update. Nature. 1989 Jul 13;340(6229):103–104. doi: 10.1038/340103a0. [DOI] [PubMed] [Google Scholar]
  36. Mandon E. C., Milla M. E., Kempner E., Hirschberg C. B. Purification of the Golgi adenosine 3'-phosphate 5'-phosphosulfate transporter, a homodimer within the membrane. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10707–10711. doi: 10.1073/pnas.91.22.10707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Marini A. M., Vissers S., Urrestarazu A., André B. Cloning and expression of the MEP1 gene encoding an ammonium transporter in Saccharomyces cerevisiae. EMBO J. 1994 Aug 1;13(15):3456–3463. doi: 10.1002/j.1460-2075.1994.tb06651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Milla M. E., Clairmont C. A., Hirschberg C. B. Reconstitution into proteoliposomes and partial purification of the Golgi apparatus membrane UDP-galactose, UDP-xylose, and UDP-glucuronic acid transport activities. J Biol Chem. 1992 Jan 5;267(1):103–107. [PubMed] [Google Scholar]
  39. Milla M. E., Hirschberg C. B. Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1786–1790. doi: 10.1073/pnas.86.6.1786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Moremen K. W., Touster O., Robbins P. W. Novel purification of the catalytic domain of Golgi alpha-mannosidase II. Characterization and comparison with the intact enzyme. J Biol Chem. 1991 Sep 5;266(25):16876–16885. [PubMed] [Google Scholar]
  41. Moremen K. W., Trimble R. B., Herscovics A. Glycosidases of the asparagine-linked oligosaccharide processing pathway. Glycobiology. 1994 Apr;4(2):113–125. doi: 10.1093/glycob/4.2.113. [DOI] [PubMed] [Google Scholar]
  42. Munro S. An investigation of the role of transmembrane domains in Golgi protein retention. EMBO J. 1995 Oct 2;14(19):4695–4704. doi: 10.1002/j.1460-2075.1995.tb00151.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ninnemann O., Jauniaux J. C., Frommer W. B. Identification of a high affinity NH4+ transporter from plants. EMBO J. 1994 Aug 1;13(15):3464–3471. doi: 10.1002/j.1460-2075.1994.tb06652.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Paulson J. C., Rogers G. N. Resialylated erythrocytes for assessment of the specificity of sialyloligosaccharide binding proteins. Methods Enzymol. 1987;138:162–168. doi: 10.1016/0076-6879(87)38013-9. [DOI] [PubMed] [Google Scholar]
  45. Pessino A., Hebert D. N., Woon C. W., Harrison S. A., Clancy B. M., Buxton J. M., Carruthers A., Czech M. P. Evidence that functional erythrocyte-type glucose transporters are oligomers. J Biol Chem. 1991 Oct 25;266(30):20213–20217. [PubMed] [Google Scholar]
  46. Roth J. Subcellular organization of glycosylation in mammalian cells. Biochim Biophys Acta. 1987 Oct 5;906(3):405–436. doi: 10.1016/0304-4157(87)90018-9. [DOI] [PubMed] [Google Scholar]
  47. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  48. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Seed B., Aruffo A. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc Natl Acad Sci U S A. 1987 May;84(10):3365–3369. doi: 10.1073/pnas.84.10.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stanley P. Glycosylation mutants of animal cells. Annu Rev Genet. 1984;18:525–552. doi: 10.1146/annurev.ge.18.120184.002521. [DOI] [PubMed] [Google Scholar]
  51. Stanley P., Sallustio S., Krag S. S., Dunn B. Lectin-resistant CHO cells: selection of seven new mutants resistant to ricin. Somat Cell Mol Genet. 1990 May;16(3):211–223. doi: 10.1007/BF01233357. [DOI] [PubMed] [Google Scholar]
  52. Stanley P., Siminovitch L. Complementation between mutants of CHO cells resistant to a variety of plant lectins. Somatic Cell Genet. 1977 Jul;3(4):391–405. doi: 10.1007/BF01542968. [DOI] [PubMed] [Google Scholar]
  53. Stanley P., Sudo T., Carver J. P. Differential involvement of cell surface sialic acid residues in wheat germ agglutinin binding to parental and wheat germ agglutinin-resistant Chinese hamster ovary cells. J Cell Biol. 1980 Apr;85(1):60–69. doi: 10.1083/jcb.85.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Takano R., Muchmore E., Dennis J. W. Sialylation and malignant potential in tumour cell glycosylation mutants. Glycobiology. 1994 Oct;4(5):665–674. doi: 10.1093/glycob/4.5.665. [DOI] [PubMed] [Google Scholar]
  55. Varki A. Selectins and other mammalian sialic acid-binding lectins. Curr Opin Cell Biol. 1992 Apr;4(2):257–266. doi: 10.1016/0955-0674(92)90041-a. [DOI] [PubMed] [Google Scholar]
  56. Verbert A., Cacan R., Cecchelli R. Membrane transport of sugar donors to the glycosylation sites. Biochimie. 1987 Feb;69(2):91–99. doi: 10.1016/0300-9084(87)90240-9. [DOI] [PubMed] [Google Scholar]
  57. Wang W. C., Cummings R. D. The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked alpha-2,3 to penultimate galactose residues. J Biol Chem. 1988 Apr 5;263(10):4576–4585. [PubMed] [Google Scholar]
  58. Wickens M., Stephenson P. Role of the conserved AAUAAA sequence: four AAUAAA point mutants prevent messenger RNA 3' end formation. Science. 1984 Nov 30;226(4678):1045–1051. doi: 10.1126/science.6208611. [DOI] [PubMed] [Google Scholar]
  59. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  60. Yogeeswaran G., Salk P. L. Metastatic potential is positively correlated with cell surface sialylation of cultured murine tumor cell lines. Science. 1981 Jun 26;212(4502):1514–1516. doi: 10.1126/science.7233237. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES